SUMMARY Cell-restricted transcriptional modulators play critical roles in the process of selective gene regulation during hematopoiesis. We have been investigating the molecular and biological function of Erythroid Krppel-like Factor (EKLF; KLF1). EKLF is a cell-restricted transcription factor that is a global regulator of genes essential for the erythroid program. Analysis of EKLF's ability to positively regulate transcription and chromatin has led to identification of its association with the HIRA chaperone, and of its direct activation of histone H3.3 expression. The experiments of Aim 1 will link a coherent feed-forward model of the EKLF/HIRA/H3.3 axis with changes in paused/active status at selected EKLF target genes. Analysis of expression patterns resulting from the neonatal anemia (Nan) mutation in murine EKLF (E339D) shows extensive genetic disruption and exacerbated anemia that follows extensive ectopic gene expression following Nan-EKLF expression. The experiments of Aim 2 will have identified alterations in protein/protein interactions, mapped the changes in binding at normal and novel target sites, and analyzed the resultant epigenetic rearrangements that follow from Nan-EKLF expression. Analysis of DNA from a congenital dyserythropoietic anemia (CDA) patient supports the idea that a subset (type IV) follows from mutation in one allele of human KLF1 at E325K. The experiments of Aim 3 will have characterized derived iPSCs and de novo mutated erythroid cells for their hematopoietic/erythroid cellular and expression capabilities as well as to enable molecular/ biochemical studies of the KLF1/E325K protein itself. These studies will be aided by the use of phenotypic and biochemical analyses, genetic approaches, and use of primary or minimally manipulated cells. Elucidating EKLF's role in regulatory phenomena will continue to illuminate novel aspects of erythroid biology and the essential mechanisms by which a cell-restricted transcription factor can exert varied yet highly controlled influences on genetic expression and epigenetics.